Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
Language English Country Great Britain, England Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
22079107
DOI
10.1016/j.phytochem.2011.10.008
PII: S0031-9422(11)00477-8
Knihovny.cz E-resources
- MeSH
- Amino Acids metabolism MeSH
- beta-Glucosidase chemistry genetics MeSH
- Cytokinins metabolism MeSH
- Glucosides metabolism MeSH
- Hydrolysis MeSH
- Isomerism MeSH
- Zea mays chemistry enzymology genetics MeSH
- Molecular Conformation MeSH
- Mutation MeSH
- Mutagenesis, Site-Directed methods MeSH
- Genes, Plant MeSH
- Plant Proteins chemistry genetics MeSH
- Amino Acid Sequence MeSH
- Substrate Specificity MeSH
- Binding Sites MeSH
- Structure-Activity Relationship MeSH
- Zeatin metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Amino Acids MeSH
- beta-Glucosidase MeSH
- Cytokinins MeSH
- Glucosides MeSH
- Plant Proteins MeSH
- Zeatin MeSH
The maize β-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-D-glucopyranoside versus the trans-zeatin-O-β-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.
References provided by Crossref.org
Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants
New Insights Into the Metabolism and Role of Cytokinin N-Glucosides in Plants
High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing
An automated method to evaluate the enzyme kinetics of β-glucosidases
Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction
